ABSTRACT
Double-stranded RNA adenosine deaminase 1 (ADAR1) is significantly down-regulated in fibroblasts derived from Idiopathic Pulmonary Fibrosis (IPF) patients, and its overexpression restored levels of miRNA-21, PELI1, and SPRY2. There are two ADAR1 isoforms in humans, ADAR1-p110 and ADAR1-p150, generated by an alternative promoter. Let-7d is considered an essential microRNA in Pulmonary Fibrosis (PF). In silico analysis revealed COL3A1 and SMAD2, proteins involved in the development of IPF, as Let-7d targets. We analyzed the role of ADAR1-p110 and ADAR1-p150 isoforms in the regulation of Let-7d maturation and the effect of this regulation on the expression of COL3A1 and SMAD2 in IPF fibroblast. We demonstrated that differential expression and subcellular distribution of ADAR1 isoforms in fibroblasts contribute to the up-regulation of pri-miR-Let-7d and down-regulation of mature Let-7d. Induction of overexpression of ADAR1 reestablishes the expression of pri-miR-Let-7d and Let-7d in lung fibroblasts. The reduction of mature Let-7d upregulates the expression of COL3A1 and SMAD2. Thus, ADAR1 isoforms and Let-7d could have a synergistic role in IPF, which is a promising explanation in the mechanisms of fibrosis development, and the regulation of both molecules could be used as a therapeutic approach in IPF.
Subject(s)
Adenosine Deaminase , Idiopathic Pulmonary Fibrosis , MicroRNAs , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Humans , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lung/metabolism , Membrane Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding ProteinsABSTRACT
Chronic obstructive pulmonary disease (COPD) is a disease with an inflammatory phenotype with increasing prevalence in the elderly. Expanded population of mutant blood cells carrying somatic mutations is termed clonal hematopoiesis of indeterminate potential (CHIP). The association between CHIP and COPD and its relevant effects on DNA methylation in aging are mainly unknown. Analyzing the deep-targeted amplicon sequencing from 125 COPD patients, we found enhanced incidence of CHIP mutations (~20%) with a predominance of DNMT3A CHIP-mediated hypomethylation of Phospholipase D Family Member 5 (PLD5), which in turn is positively correlated with increased levels of glycerol phosphocholine, pro-inflammatory cytokines, and deteriorating lung function.
Subject(s)
Clonal Hematopoiesis , Pulmonary Disease, Chronic Obstructive , Aged , Gene Expression , Hematopoiesis/genetics , Humans , Mutation/genetics , Pulmonary Disease, Chronic Obstructive/geneticsABSTRACT
INTRODUCTION: microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. METHODS: Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFß1. Real-time PCR and Western blot were performed. RESULTS: ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. CONCLUSION: These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.
Subject(s)
Adenosine Deaminase/metabolism , Fibroblasts/enzymology , Idiopathic Pulmonary Fibrosis/enzymology , Lung/enzymology , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , Adenosine Deaminase/genetics , Case-Control Studies , Cells, Cultured , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Expression Regulation, Enzymologic , Humans , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/pathology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lung/drug effects , Lung/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Primary Cell Culture , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/genetics , Transforming Growth Factor beta1/pharmacology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
OBJECTIVE: The objective of this study is to determine the effect of two angiotensin-converting enzyme inhibitors (ACEi) (Enalapril and Captopril), an angiotensin-II receptor inhibitor (Losartan) and a renin inhibitor (Aliskiren) on renin, TGF-ß1 and collagen expressions in human lung fibroblast cultures through real-time PCR and ELISA. MATERIALS AND METHODS: Normal commercial fibroblasts (CCD25) were exposed to 10(-6) M of enalapril, captopril, losartan, or aliskiren for 6 h. Subsequently, media were recovered and proteins were concentrated; RNA was extracted from the cells. Real time-PCR and ELISA were performed. RESULTS: ACEi and losartan-stimulated fibroblasts showed an increase in the expression of TGF-ß1, Collagen-Iα1 (Col-Iα1), and renin (except losartan) vs PolR2A (p < 0.05), and upregulation of TGF-ß1 protein (p < 0.01), except with aliskiren. CONCLUSION: Results show that ACEis and losartan could play a profibrosing role by inducing the overexpression of molecules such TGF-ß1 and Collagen.
